Influenza B Virus BM2 Protein Has Ion Channel Activity that Conducts Protons across Membranes

Mould, Jorgen; Paterson, Reay G.; Takeda, Makoto; Ohigashi, Yuki; Venkataraman, Padma; Lamb, Robert A.; Pinto, Lawrence H.

doi:10.1016/s1534-5807(03)00190-4

Cited by 142 publications

(163 citation statements)

References 43 publications

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“…Although this finding is consistent with our knowledge about the A/M2 oligomeric state (15) (23). BM2 channels allow proton flow upon the acidification of the extracellular medium (4). Substitution of the pore lining residue His-19 with Cys (BM2(H19C)) results in the formation of non-functional channels (4).…”

Section: Bm2 Can Be Chemically Cross-linked To the Tetramericsupporting

confidence: 88%

“…RNA segment 7 of both influenza A and B viruses encodes the M2 integral membrane protein (A/M2 and BM2, respectively) that functions as a pH-sensitive proton channel and is essential for virus replication (1)(2)(3)(4)(5). A/M2 and BM2 proteins both consist of a small N-terminal ectodomain (24 and 7 residues, respectively), a single transmembrane domain, 19 amino acids long, and a cytoplasmic tail (54 and 83 residues, respectively) (5).…”

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confidence: 99%

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The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Balannik

Lamb

Pinto

2008

Journal of Biological Chemistry

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Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.Influenza A and B viruses are enveloped viruses with a genome consisting of eight segments of negative-strand RNA. RNA segment 7 of both influenza A and B viruses encodes the M2 integral membrane protein (A/M2 and BM2, respectively) that functions as a pH-sensitive proton channel and is essential for virus replication (1-5). A/M2 and BM2 proteins both consist of a small N-terminal ectodomain (24 and 7 residues, respectively), a single transmembrane domain, 19 amino acids long, and a cytoplasmic tail (54 and 83 residues, respectively) (5). Although A/M2 and BM2 proteins have similar structural and functional properties, the only homology between their amino acid sequences is found in the HXXXW motif of the membrane-spanning region. This motif plays a critical role in the ion channel activity (4, 6 -8). The A/M2 channel activity is inhibited by the antiviral drug amantadine (9, 10) but unfortunately, amantadine does not inhibit the BM2 channel activity (5, 11).The oligomeric state of the A/M2 channel had been investigated by several approaches. First, A/M2 was shown to form homo-oligomers, most likely homotetramers, by cross-linking and sedimentation experiments (12, 13). Site-specific mutagenesis studies demonstrated that the oligomeric structure of A/M2 channels is stabilized by disulfide bonds (14). Finally, by functional analysis of mixed oligomers of known composition, the active oligomeric form of A/M2 protein was shown to be a tetramer (15). Most recently the atomic stru...

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Section: Bm2 Can Be Chemically Cross-linked To the Tetramericsupporting

confidence: 88%

mentioning

confidence: 99%

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Balannik

Lamb

Pinto

2008

Journal of Biological Chemistry

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“…The substitution by a bulky side chain may significantly reduce the amantadine binding affinity, 8 which in turn may explain the inability of amantadine to inhibit the M2 channel activity and replication of influenza B virus. 27 In summary, our molecular dynamics simulations suggest that the tetrad of V27 forms a secondary gate. When amantadine is bound, an extended blockage is formed, leading to breakup of the water wire and inactivation of the proton channel.…”

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confidence: 70%

A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

2008

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The mechanism of inhibition of the influenza A virus M2 proton channel by the antiviral drug amantadine has been under intense investigation. The importance of a mechanistic understanding is heightened by the prevalence of amantadine-resistant mutations. To gain mechanistic insight at the molecular level, we carried out extensive molecular dynamics simulations of the tetrameric M2 proton channel in both apo and amantadine-bound forms in a lipid bilayer. The simulation of the apo form revealed that Val27 from the four M2 subunits can form a secondary gate near the channel entrance and break the water wire in the channel pore. This gate arises from physical occlusion and the elimination of hydrogen-bonding partners for water molecules. In the presence of amantadine, the secondary gate formed by Val27 and the drug molecule lying just below form an extended blockage, which breaks the water wire throughout the simulation. The location and orientation of amantadine inside of the channel pore as found in our simulation are supported by a host of experimental observations. Our study suggests a novel role for Val27 in the inhibition of the M2 proton channel by amantadine.The M2 protein of the influenza A virus is a tetrameric proton-selective ion channel activated by low pH, and its channel activity is essential for the life cycle of the virus. The antiviral drug amantadine inhibits the replication of the virus by putatively binding to the transmembrane domain (TMD) of the M2 proton channel. 1 However, over 90% of recent influenza A cases were found to have the S31N mutation on the M2 protein that confers amantadine resistance. 2 Along with experiments, 3-9 extensive computational studies [10][11][12][13][14][15][16][17] have been performed to model the structure of the M2 TMD and to understand the mechanisms of conductance and selectivity of the proton channel. The tetrad of H37 is part of the putative primary gate essential for channel conductance and selectivity. 5,9 The structure of M2 TMD when amantadine is present has been determined recently by solidstate NMR spectroscopy. 18 Here, we report a study aimed at modeling the binding of amantadine to M2 TMD. Our results present both mechanistic insight on the inhibition of M2 by amantadine and possible explanations for mutations leading to amantadine resistance.We modeled an amantadine molecule into the structure of M2 TMD determined in the presence of amantadine (PDB code 2h95; 18 see "Setup of Simulation Systems" and Figure S1 in Supporting Information). Amantadine was initially positioned around S31, in line with NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the central location of this residue in the constellation of amantadine-resistant mutations (on V27, A30, S31, and G34). 3 Parallel molecular dynamics simulations were then carried out for up to 15 ns on M2 TMD in the amantadine-bound form and in the apo form (the latter based on the apo structure found in PDB code 1nyj 19 ).We monitored the radii of the channel pores across...

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“…There are, however, significant differences between the two channels. BM2 channel activity is higher than that of AM2 (Mould et al, 2003;Wang et al, 2009), and, unlike AM2, the BM2 proton conductance is completely insensitive to amantadine and rimantadine (Mould et al, 2003). In fact, small molecule inhibitors that block the BM2 channel have not yet been identified.…”

Section: Introductionmentioning

confidence: 99%

“…After BM2 is synthesized in the infected host cell, it is incorporated into the TGN membrane and transported to cell surface for virus budding (Watanabe et al, 2003). BM2 proton conductance has a pH profile similar to that of AM2 (Mould et al, 2003). There are, however, significant differences between the two channels.…”

Section: Introductionmentioning

confidence: 99%

Flu channel drug resistance: a tale of two sites

Pielak

Chou

2010

Protein Cell

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The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.

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Influenza B Virus BM2 Protein Has Ion Channel Activity that Conducts Protons across Membranes

Cited by 142 publications

References 43 publications

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

Flu channel drug resistance: a tale of two sites

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